Chemical and enzymatic reactions are used to detect or quantitate the presence of certain substances in microbiological or other assays. Many of these tests rely on the development or change of color or fluorescence to indicate the presence or quantity of the substance of interest.
There are many examples of reactions used in Microbiology that rely on a color change. Bascomb, Enzyme Tests in Bacterial Identification, Meth. Microbiol. 19, 105 (1987). For example, a variety of organisms can be classified in large part by their pattern of fermentation, oxidation or assimilation of carbon sources. Fermentation of carbohydrates results in the production of acid which causes a decrease in pH. This drop in pH can be easily detected by including a pH indicator like bromothymol blue or phenol red. With both indicators, acid conditions representing the fermentation of a particular carbohydrate result in a yellow color (changing from blue-green for bromothymol blue or pink/red for phenol red). The same approach can be adopted for a variety of carbohydrates, ranging from monosaccharides like glucose to polysaccharides like inulin. In an analogous fashion, increasing pH can also be followed. Assays for detecting the presence of decarboxylase and urease, and the ability to use malonate are based on an increase in pH, as indicated by a color change in an indicator.
Another approach to determine if an organism can degrade a particular substrate, is to use a reagent which is capable of reacting with one or more of the intermediates or final products. For example, the detection of the reduction of nitrate to nitrite. If nitrite is formed, then a pink to deep red color will result when sulfanilic acid and alpha-napthylamine are added to the reaction mixture.
In contrast to the indirect detection of an enzymatic reaction illustrated by the nitrate/nitrite test, it is possible to use a synthetic analog of a natural substrate to directly indicate the presence of an enzyme. For example, methylene blue can be reduced under certain conditions by the action of reductase, resulting in a shift from blue to colorless. In another test, the oxidase assay relies on the interaction of cytochrome oxidase with N,N,N',N'-tetramethyl-p-phenylenediamine producing a blue color.
Another example is the ability of microorganisms to degrade sulfur-containing amino acids as indicated by the production of H.sub.2 S. Typically, the organism is incubated with a high concentration of a sulfur-containing substrate (e.g. cysteine, cystine) in an acid environment. The production of H.sub.2 S is indicated by the formation of a black precipitate in the presence of ferric ammonium citrate.
Enzymes can usually act on more than one substrate. This allows for the use of synthetic enzyme substrates for the detection of enzyme activities. Synthetic substrates contain a metabolic moiety conjugated with a chromatic or fluorescent moiety. The conjugated molecule usually has a different absorption and/or emission spectrum from the unconjugated form. Moreover, the unconjugated chromatic or fluorescent moiety shows a considerably higher absorption or fluorescence coefficients than those of the conjugated molecule. This allows the measurement of small amounts of products of enzyme activities in the presence of the large amounts of conjugated substrate required for maximal enzyme activity. An example of a synthetic enzyme substrate is o-nitro-phenol-.beta.-galactopyranoside used for the detection of activity of the enzyme .beta.-galactosidase. The conjugated substrate is colorless. The .beta.-galactosidase enzyme hydrolyzes the substrate to yield .beta.-galactosidase and o-nitro phenol. o-nitro-phenol absorbs strongly at 405 nm, and its release can be measured by the increase in absorbance at that wavelength. Bascomb, Enzyme Tests in Bacterial Identification, Meth. Microbiol. 19, 105 (1987), reviewed the synthetic moieties used for enzyme substrates and the enzymatic activities measurable using this principle.
Presently, the monitoring of color or color end-product in chemical and microbial reactions is usually achieved in either of two ways; 1) the detection of color or color end-product can be achieved by visual observation and estimated qualitatively, or 2) the detection of color end-products or loss of color can be achieved by measuring the intensity of color instrumentally. Spectrophotometers that measure light absorbance are commonly used for this purpose. When measuring the concentration of a number of substances it is advantageous to use one instrument or one principle of measurement, otherwise cost is increased.
Although the use of colorimetric reactions is widespread there are limitations, especially in the sensitivity of detection. In order to improve sensitivity and, in the case of identification of microorganisms, thereby to decrease the time required to obtain a result, fluorescence-based methods frequently are used. Unfortunately, it may not be possible to develop a fluorescent equivalent to every assay. Additionally, the fluorescent reagents themselves may be highly toxic and therefore difficult to commercialize.
In such cases one might need to measure activities of some enzymes fluorometrically, the others colorimetrically. However, most instruments are suited to measure either absorbance or fluroescence, and very few can be used to measure both.
The general principle of fluorescence quenching has been accepted as a way to detect or determine enzymatic or chemical reactions. For example, Fleminger et al. synthesized intramolecularly quenched fluorogenic substrates for the assay of bacterial aminopeptidase P. Fleminger et al., Fluorogenic Substrates for Bacterial Aminopeptidase P and its Analogs Detected in Human Serum and Calf Lung, Eur. J. Biochem. 125, 609 (1982). In this case, the fluorescence of the aminobenzoyl group is quenched by the presence of a nitrophenylalanyl group. When the enzyme is present, the nitrophenylalanyl group is cleaved, with a concommitant increase in the sample's fluorescence. A variety of enzymes have been assayed by this type of procedure, including hydrolytic enzymes, other amino- and carboxypeptidases and an endopeptidase. Yaron et al., Intramolecularly Quenched Fluorogenic Substrates for Hydrolytic Enzymes, Anal. Biochem. 95, 228 (1979); Carmel et al., Intramolecularly- Quenched Fluorescent Peptides as Fluorogenic Substrates of Leucine Aminopeptidase and Inhibitors of Clostridial Aminopeptidase, Eur. J. Biochem. 73, 617 (1977); Carmel et al., An Intramolecularly Quenched Fluorescent Tripeptide as a Fluorogenic Substrate of Angiotensin-I-Converting Enzyme and of Bacterial Dipeptidyl Carboxypeptidase, Eur. J. Biochem. 87, 265 (1978); Florentin et al., A Highly Sensitive Fluorometric Assay for "Enkephalinase", a Neutral Metalloendopeptidase that Releases Tyrosine-Glycine-Glycine from Enkephalins, Anal. Biochem 141, 62 (1984). In each of the previous approaches, a synthetic substrate containing a quenching group and a fluorescing group was generated in order to detect the activity of the enzyme.
An alternative to this approach would involve the synthesis of a resonance energy transfer pair of fluorescing groups on a substrate molecule. In this method, cleavage by the enzyme of one of the groups would result in a decrease in fluorescence, since the critical distance would be exceeded, eliminating the transfer of energy. However, the previously discussed approaches are limited to specifically designed substrates.
Still another approach involves the estimation of a chromophore by fluorescence measurement. See W. Blumberg et al., Hemoglobin Determined in Whole Blood "Front Face" Fluorometry, Clin. Hemo. 26, 409 (1980). Blumberg disclosed an assay based on attenuation of fluorescence of a dye, whose excitation wavelengths overlap with the absorption wavelengths of the chromophore.
Subsequently, M. Shaffer, U.S. Pat. No. 4,495,293 (hereinafter Shaffer) filed a patent application disclosing a method to fluorometrically determine a ligand in an assay solution using conventional fluorometric techniques. In Shaffer the intensity of the fluorescence emitted by the assay solution is related to the change in transmissive properties of the assay solution produced by the interaction of the ligand to be determined and a reagent system capable of producing change in the transmissive properties of the assay solution in the presence of the ligand. More particularly, Shaffer discloses a method to monitor absorbance using a fluorophore in solution with the chromophore. In this method the fluorophore may interact with the assay cocktail and produce changes in fluorescence intensity which are unrelated to the change being measured. The selection of the fluorophores is also restricted, in that pH dependent or environment sensitive fluorophores cannot be utilized. Additionally, when the fluorophore is in solution, less than accurate measure of absorbance may be obtained because light is absorbed exponentially through the chromophore sample.
Similarly, Beggs & Sand, EPA 91,837 disclosed a solution based method for determination of tryptophan-deaminase activity by measuring the reduction in fluorescence in the presence of a chromophore produced by the interaction between indole pyruvic acid and metal ions using a fluorophore "whose fluorescence is capable of being quenched by the indole pyruvate-metal ion complex, the ions of the fluorophore being present throughout the incubation period".
Also, Sands, U.S. Pat. No. 4,798,788 discloses a process to detect a nitrate reducing microorganism by measuring reduction of fluorescence in solution by causing the diazotization of the fluorophore. In all these cases a specific fluorophore needs to be chosen for each test to ensure that it will fluoresce under the conditions of the test, e.g. only few fluorophores fluoresce at pH of less than 2.0.
Consequently, a need exists to develop a general process to detect or determine the concentration of any substance so that any fluorophore with the appropriate spectral characteristics may be employed. Additionally, a need exists to develop a process to maximize the amount of light detected in a fluorometric assay so that assay sensitivity can be increased.